Sequencing SARS-CoV-2 from Animal Samples with NEBNext® ARTIC SARS-CoV-2 Companion Kit
Isabel Gautreau, Brianna Stenger
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Abstract
This protocol is adapted for animal samples and is based on the methods for the NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®), NEB #E7660S/L 24/96 reactions.
This protocol does not include a cleanup and normalization step for each sample after cDNA synthesis. Performing the cleanup and normalization step creates library pools where the reads for each library are more evenly distributed. Skipping these steps reduces hands on time but may require a longer sequencing run to obtain sufficient coverage for each sample. To obtain more even sample to sample coverage, we recommend normalizing the RNA samples prior to starting the protocol.
Changes in this version:
- Removal of the human primers
- Combining multiple barcoding reactions (step 15 and 21)
- Addition of Loading Flongle Flow Cell section
- Addition of Analysis section
Before start
Note: The amount of RNA required for detection depends on the abundance of the RNA of interest. In general, we recommend, using > 10 copies of the (SARS-CoV-2) viral genome as input. In addition, we recommend setting up a no template control reaction and all reactions are set-up in a hood .
Note: : If sample Ct is between 12-15, then it is recommended per the nCoV 2019 sequencing protocol v3 LoCost to dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR inhibition.
Steps
cDNA Synthesis
The presence of genomic DNA or carry-over products can interfere with sequencing accuracy, particularly for low copy targets. Therefore, it is important to carry out the appropriate no template control (NTC) reactions to demonstrate that positive reactions are meaningful.
Gently mix 10 times by pipetting and spin down the LunaScript RT SuperMix reagents (contains primers). Prepare the cDNA synthesis reaction as described below:
| A | B |
|---|---|
| COMPONENT | VOLUME |
| RNA Sample* | 8 µl |
| (lilac) LunaScript RT SuperMix | 2 µl |
| Total Volume | 10 µl |
*Up to 0.5 µg total RNA can be used in a 10 µl reaction.
Flick the tube or pipet up and down 10 times to mix followed by a quick spin.
For no template controls, mix the following components:
| A | B |
|---|---|
| COMPONENT | VOLUME |
| (white) Nuclease-free Water | 8 µl |
| (lilac) LunaScript RT SuperMix | 2 µl |
| Total Volume | 10 µl |
Flick the tube or pipet up and down 10 times to mix followed by a quick spin.
Incubate reactions in a thermocycler with lid temperature at 105°C with the following steps:
| A | B | C | D |
|---|---|---|---|
| CYCLE STEP | TEMP | TIME | CYCLE |
| Primer Annealing | 25°C | 2 minutes | 1 |
| cDNA Synthesis | 55°C | 20 minutes | |
| Heat Inactivation | 95°C | 1 minute |
Targeted cDNA Amplification
Gently mix Q5 Hot Start High Fidelity 2X Master Mix 10 times by pipetting and spin down reagents. Prepare the split pool amplification reactions as described below:
For Pool Set A: .
| A | B |
|---|---|
| COMPONENT | VOLUME |
| cDNA (Step 6) | 4.5 µl |
| (lilac) Q5 Hot Start High-Fidelity 2X Master Mix | 6.25 µl |
| NEBNext ARTIC SARS-CoV-2 Primer Mix 1* | 1.75 µl |
| Total Volume | 12.5 µl |
For Pool Set B:
| A | B |
|---|---|
| COMPONENT | VOLUME |
| cDNA (Step 6) | 4.5 µl |
| (lilac) Q5 Hot Start High-Fidelity 2X MM | 6.25 µl |
| NEBNext ARTIC SARS-CoV-2 Primer Mix 2* | 1.75 µl |
| Total Volume | 12.5 µl |
Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.
Incubate reactions in a thermocycler** with the following steps:
| A | B | C | D |
|---|---|---|---|
| CYCLE STEP | TEMP | TIME | CYCLES |
| Initial Denaturation | 98°C | 30 seconds | 1 |
| Denature | 95°C | 15 seconds | 35 |
| Annealing/Extension | 63°C* | 5 minutes | |
| Hold | 4°C | ∞ | 1 |
- It is very important to set up the annealing and extension temperature to 63°C** Set heated lid to 105°C.
PCR Reaction Pooling
For each sample, combine pool A and pool B PCR Reactions.
NEBNext End Prep
Add the following components to a PCR tube (End Prep Reaction and Buffer can be pre-mixed and master mix is stable On ice for 4 hours):
| A | B |
|---|---|
| COMPONENT | VOLUME |
| Targeted cDNA Amplicons (Step 11) | 1 µl |
| (white) Nuclease-free water | 11.5 µl |
| (green) NEBNext Ultra II End Prep Reaction Buffer | 1.75 µl |
| (green) NEBNext Ultra II End Prep Enzyme Mix | 0.75 µl |
| Total Volume | 15 µl |
Flick the tube or pipet up and down 10 times to mix the solution. Perform a quick spin to collect all liquid from the sides of the tube.
Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:
0h 10m 0s @ 20°C
0h 10m 0s @ 65°C
Hold at 4°C
Barcode Ligation
Add the following components directly to a sterile nuclease-free PCR tube:
| A | B |
|---|---|
| COMPONENT | VOLUME |
| single | |
| (white) Nuclease-free water | 6 µl |
| End-prepped DNA (Previous Step) | 1.5 µl |
| Native Barcode* | 2.5 µl |
| (red) Blunt/TA Ligase Master Mix** | 10 µl |
| Total | 20 µl*** |
- Native Barcodes are provided in Oxford Nanopore Technologies Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) or 1-96 (EXP-NBD196)** Mix the Blunt/TA Ligase Master Mix by pipetting up and down several times prior to adding to the reaction.*** Multiple ligation reactions for a sample with the same barcode can be pooled (see #21) to increase the amount of product for cleanup. Recommend separate tubes for individual 20uL reactions and then combine in step 21. Unsure how a 40uL reaction will respond to the cycling conditions.
Flick the tube or pipet up and down 10 times to mix solution. Perform a quick spin to collect all liquid from the sides of the tube.
Incubate at 25°C for 0h 20m 0s.
Incubate at 65°C for 0h 10m 0s.
Place 65On ice for 0h 1m 0s.
Pool all barcoded samples into one 1.5 ml DNA LoBind Tube.
Cleanup of Barcoded DNA
Vortex NEBNext Sample Purification Beads to resuspend.
Add 0.4X resuspended beads to pooled, barcoded samples (Step 20)
There is a maximum of 480uL of pooled samples. So multiple barcoded samples can be pooled.
As an example, for seven samples, including four duplicated samples:
40+40+40+40+20+20+20uL = 220µL with 88µL of resuspended Sample Purification beads
Mix well by flicking the tube or pipetting up and down 10 times. Perform a quick spin for 0h 0m 1s to collect all liquid from the sides of the tube.
Incubate samples on bench top for 0h 10m 0s at 65Room temperature.
Place the tube on a 1.5 ml magnetic stand (such as NEB S1506) to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
After 0h 3m 0s (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Wash the beads by adding 250µL. Flick the tube or pipet up and down 10 times to mix to resuspend pellet. If necessary, quickly spin the sample for 0h 0m 1s to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
Place the tube on an appropriate magnetic stand for 0h 3m 0s 3 minutes (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
Repeat Step 26 and 27 once for a total of two washes:
Wash the beads by adding 250µL. Flick the tube or pipet up and down to mix to resuspend pellet. If necessary, quickly spin the sample for 0h 0m 3s to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
Place the tube on an appropriate magnetic stand for 0h 3m 0s (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube, place back on the magnetic stand and remove traces of SFB with a p10 pipette tip.
Add 500µL to the tube while on the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Repeat the previous step once to make it a total of 2 washes.
Perform a quick spin and place the sample tube on the magnetic stand, remove any residual ethanol.
Air dry the beads for 0h 0m 30s while the tube is on the magnetic stand with the lid open.
Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 33µL.
Resuspend the pellet by flicking the tube or pipetting up and down 10 times to mix. Incubate for at least 2 minutes at Room temperature. If necessary, quickly spin the sample for 0h 0m 1s to collect the liquid from the sides of the tube before placing back on the magnetic stand.
Place the tube on the magnetic stand. After 2 minutes (or when the solution is clear), transfer 32µL to a new 1.5 ml Eppendorf DNA LoBind Tube or PCR tube.
Assess the concentration of DNA targets using 1µL of sample with a DeNovix with FX fluorometer module. Use
It is also recommend using a Qubit fluorometer for concentration assessment. (Nanodrop is NOT recommended since it may overestimate the DNA concentration).
Adapter Ligation
Use the Qubit readings from the previous Step to dilute purified Native barcoded DNA pool in nuclease-free water. Dilute the sample to a concentration of 60 ng total in a final volume of 30 μl (or 2 ng/µl in 30 µl). Add the following components into a 1.5 ml Eppendorf DNA LoBind Tube or nuclease-free PCR tube:
| A | B |
|---|---|
| COMPONENT | VOLUME |
| Native barcoded and purified DNA (Step 35, up to 60 ng) | 30 µl |
| (red) NEBNext Quick Ligation Reaction Buffer * | 10 µl |
| Adapter Mix II (AMII)** | 5 µl |
| (red) NEBNext Quick T4 Ligase | 5 µl |
| Total Volume | 50 µl |
- Mix the NEBNext Quick Ligation Reaction Buffer by pipetting up and down several times prior to adding to the reaction. ** Adapter Mix II is provided by Oxford Nanopore Technologies Native Barcoding Expansion 1-12 (EXP-NBD104), 13-24 (EXP-NBD114) and 1-96 (EXP-NBD-196) kits.
Flick the tube to mix solution. Perform a quick spin for 0h 0m 1s to collect all liquid from the sides of the tube.
Incubate at 25°C for 0h 20m 0s.
Proceed to Cleanup of Adapter-ligated DNA in the next section.
Cleanup of Adapter Ligated DNA
Vortex NEBNext Sample Purification Beads to resuspend.
Add 50µL to the ligation mix. Mix well by flicking the tube followed by a quick spin for 0h 0m 3s.
Incubate samples for 0h 10m 0s at 20Room temperature.
Place the tube on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
After 3 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Wash the beads by adding 250µL. Flick the tube to resuspend pellet. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand. Place the tube on an appropriate magnetic stand.
Wait for 0h 3m 0s (or until the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
Repeat Step 46 and 47 once for a total of two washes:
Wash the beads by adding 250µL. Flick the tube or pipet up and down to mix to resuspend pellet. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand. Place the tube on an appropriate magnetic stand.
Wait for 3 minutes (or when the solution is clear) to separate the beads from the supernatant. Remove the supernatant.
Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of SFB with a p10 pipette tip.
Remove the tube from the magnetic stand. Elute the DNA target from the beads by adding 15µL provided in SQK-LSK109 kit from Oxford Nanopore.
Resuspend the pellet well in EB buffer by flicking . Incubate for 0h 10m 0s at 20Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
Place the tube/plate on the magnetic stand. After 0h 3m 0s (or when the solution is clear), transfer 15µL to a new DNA LoBind tube.
Determine DNA concentration by quantifying 1µL.
Loading Flongle Flow Cell
Follow Oxford Nanopore Protocol SQK-LSK109 to prepare MinION® flow cell and DNA library sequencing mix using up to 20 ng adapter-ligated DNA sample (previous step).
Combine 117µLof Flush Buffer + 3µL Flush Tether in a LoBind tube and mix by pipetting
Prime the flongle flow cell by slowly pipetting the majority of the previous mix, avoid pushing too fast and bubbles
In another LoBind tube, combine 15µL of Sequencing Buffer II + 10µLof freshly vortexed Loading Beads II +5µLof ~20fmol DNA library (if volume was less than 5uL, bring up to 5uL with Elution Buffer.
Load ~30uL on the flongle flow cell, avoid bubbles.
Analysis
Used the EPI2ME Fastq + ARTIC + NextClade workflow for analyses.
